Summary Cryptosporidium is a common causative agent of chronic diarrheal disease in immunocompromised patients, such as those with HIV/AIDS, and in young children, particularly in the developing world. The majority of human infections are caused by C. parvum, which is often acquired from animals, and C. hominis, which is mainly transmitted human-to-human. Cryptosporidium infection is primarily concentrated in the small intestine, where the parasite resides within a specialized vacuole that sits atop an intestinal epithelial cell, protruding into the lumen of the intestine. Although this vacuole shows highly developed ultrastructural features, such as an actin-rich pedestal in the host cell and a membranous feeder organelle within the parasite, very little is known about the proteins that function at this host-pathogen interface. Progress in studying the biology of Cryptosporidium has been hampered by the lack of small animal models and in vitro systems for long-term cultivation. Fortunately, the latter barrier has recently been solved by the development of robust in vitro systems for cultivation. In preliminary studies, we have shown that stem cell-derived cultures of primary mouse Intestinal Epithelial Cells (mIECs) allow complete development and long- term culture of C. parvum in vitro. In mIEC cultures, C. parvum undergoes both asexual and sexual phases of development, culminating in production of oocysts. We have used this system to develop a panel of monoclonal antibodies (mAb) that define novel Cryptosporidium antigens expressed during intracellular development. In this discovery-focused project, we will explore several of these mAbs that recognize antigens that localize to the feeder organelle in trophozoites or that are found within apical secretory compartments. We will utilize these mAbs to immunoprecipitate C. parvum antigens and perform mass spectrometry to identify the proteins they recognize. We will leverage recent advances in CRISPR/Cas9 technology to epitope- tag the genes encoding these antigens and generate transgenic parasite lines. Finally, we will use these exemplary proteins for permissive biotin labeling to define the parasite secretome and characterize proteins that are concentrated at the host-pathogen interface. These exploratory studies will help define key C. parvum proteins that engage host cell receptors during invasion or that interact with host cellular pathways during intracellular development.